Fungal and Host Transcriptome Analysis of Ph-Regulated Genes

Barad et al. BMC Genomics (2016) 17:330 DOI 10.1186/s12864-016-2665-7

RESEARCH ARTICLE Open Access Fungal and host transcriptome analysis of pH-regulated genes during colonization of apple fruits by Penicillium expansum Shiri Barad1,2†,NoaSela3†, Dilip Kumar1, Amit Kumar-Dubey1, Nofar Glam-Matana1,2,AmirSherman4 and Dov Prusky1*

Abstract Background: Penicillium expansum is a destructive phytopathogen that causes decay in deciduous fruits during postharvest handling and storage. During colonization the fungus secretes D-gluconic acid (GLA), which modulates environmental pH and regulates mycotoxin accumulation in colonized tissue. Till now no transcriptomic analysis has addressed the specific contribution of the pathogen's pH regulation to the P. expansum colonization process. For this purpose total RNA from the leading edge of P. expansum-colonized apple tissue of cv. 'Golden Delicious' and from fungal cultures grown under pH 4 or 7 were sequenced and their gene expression patterns were compared. Results: We present a large-scale analysis of the transcriptome data of P. expansum and apple response to fungal colonization. The fungal analysis revealed nine different clusters of gene expression patterns that were divided among three major groups in which the colonized tissue showed, respectively: (i) differing transcript expression patterns between mycelial growth at pH 4 and pH 7; (ii) similar transcript expression patterns of mycelial growth at pH 4; and (iii) similar transcript expression patterns of mycelial growth at pH 7. Each group was functionally characterized in order to decipher genes that are important for pH regulation and also for colonization of apple fruits by Penicillium. Furthermore, comparison of gene expression of healthy apple tissue with that of colonized tissue showed that differentially expressed genes revealed up-regulation of the jasmonic acid and mevalonate pathways, and also down-regulation of the glycogen and starch biosynthesis pathways. Conclusions: Overall, we identified important genes and functionalities of P. expansum that were controlled by the environmental pH. Differential expression patterns of genes belonging to the same gene family suggest that genes were selectively activated according to their optimal environmental conditions (pH, in vitro or in vivo) to enable the fungus to cope with varying conditions and to make optimal use of available enzymes. Comparison between the activation of the colonized host's gene responses by alkalizing Colletotrichum gloeosporioides and acidifying P. expansum pathogens indicated similar gene response patterns, but stronger responses to P. expansum, suggesting the importance of acidification by P. expansum as a factor in its increased aggressiveness. Keywords: Transcription profiling, Penicillium expansum,RNA-seq,Geneexpression,pHregulation,Pathogenicity, pH-regulated genes, Fungal genes regulated by pH, Apple genes regulated by pH

* Correspondence: [email protected] †Equal contributors 1Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, the Volcani Center, Bet Dagan 50250, Israel Full list of author information is available at the end of the article

© 2016 Barad et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Barad et al. BMC Genomics (2016) 17:330 Page 2 of 27

Background resident microorganisms [5] have suggested a wide var- The genus Penicillium comprises a group of anamorphic iety of genes that may contribute to the pathogenicity of fungi in the division Ascomycota [1]. Some Penicillium this pathogen. However, the work of Barad et al. [8] has species are of economic importance because they are attributed to the pH regulation of the host by P. expan- postharvest pathogens that cause spoilage in tropical and sum specific importance for pathogenicity. In the present deciduous fruits. Penicillium expansum is the causal agent study we used a transcriptomic approach to analyze the of blue mold, which is considered one of the most import- effect of pH on fungal gene regulation and host re- ant postharvest pathogens [2] and that causes decay in de- sponses to various pH-modulating pathogens. The effect ciduous fruits during postharvest handling and storage. of the fungal response was observed by comparing the Penicillium expansum causes extensive maceration of responses of the apple to colonization by P. expansum the infected tissue by means of a common mechanism as driven by expression of the fungal gene at pH 4 or 7. of tissue acidification [3]; it acidifies its host by activation Our RNA-Seq data of fungal responses revealed nine dif- of glucose oxidase 2 (gox2) and the catalyzed oxidation of ferentially co-expressed gene clusters; they showed alter- glucose resulting in secretion of small molecules such as ations of expression patterns that were associated with D-gluconic acid (GLA) [3, 4], which modulate the envir- differential pH responses, and also with genes related to onmental pH and thereby activate several polygalacturo- P. expansum colonization. The apple response to pH nases that contribute to pectin depolymerization and was elucidated by using qRT-PCR to analyze expressions consequently tissue maceration, at pH 3.5-4 [3, 5, 6]. of selected genes in response to acidifying and alkalinizing Functional analysis of glucose oxidase 2-RNAi mutants pathogens: P. expansum and Colletotrichum gloeospor- showed a strong effect on pathogen interactions with their ioides, respectively. This analysis indicated similar patterns host: the greater the down-regulation of gox2, the stronger of host response to the respective pathogens, but the apple the impairment of GLA production, medium acidification, genes showed an extensive multifold greater response to in- and apple fruit colonization [7]. More recently, Barad et al. fection by its natural pathogen P. expansum than to that by [8] reported that P. expansum secreted not only GLA but C. gloeosporioides. This may indicate the importance of the also ammonia at the leading edge of colonization in apple acidifying modulation of pH by Penicillium as a factor for fruits. Growth of the fungus in high-sucrose media induced greater aggressiveness of P. expansum. rapid metabolism of sugar and increased GLA accumulation, and the decrease in available carbon reserves resulted in enhanced ammonia accumulation, probably because of Results and Discussion amino acid catabolism, such as occurs with other pathogens Profiling the expression of P. expansum genes during [9, 10]. These results indicated that bidirectional pH colonization and growth at pH 4 and 7 regulation occurs in P. expansum-probably dependent Genomes of P. expansum [7] were sequenced by using on nutrient availability. The question concerns which paired-end reads by means of Illumina Hiseq 2500 [12]. For metabolic processes are modulated by the pH level dur- analysis of the P. expansum transcriptomewedownloaded ing colonization of apple fruits. the reference draft genome accession JQFX00000000.1 PacC is a transcription factor that activates genes of from the Genbank site [13]. Ten libraries of single-end Aspergillus nidulans during the alkalization of the envir- RNAseq (deposited in Genbank under accession SRP071104) onment [11]. In a recent report [8], it was suggested that were mapped to the reference genome by using the P. expansum's pacC gene was active also under acidic Bowtie2 software [14]. The 10 libraries contained the conditions, and that ammonia produced at the leading following features: (i) P. expansum grown in culture media edge of the decaying tissue under low-pH conditions fur- at pH 4 for 3 h as well as a pooled sample collected at sev- ther contributed to the activation of pacC responsiveness. eral time points-0.5, 1, 3, 10, and 24 h-with two replicates This may indicate that P. expansum uses ammonia accu- at each point; (ii) P. expansum grown in culture media at mulation during nutritional modification of the ambient pH 7 for 3 h, as well as a pool collected at several time environmental pH as a regulatory cue for activation of points-0.5, 1, 3, 10, and 24 h-with two replicates at each pacC, by signaling and activating alkaline-induced genes point; and (iii) the leading edge, comprising 1–2mmof that contribute to pathogenicity and accumulation of sec- apple cv. 'Golden Delicious' tissue, colonized by P. ondary metabolites such as patulin [8]. This pH modulation expansum. For in vitro experiments, 106 spores of P. under the influence of GLA and ammonia accumulation is expansum were inoculated into germinating minimal important for understanding the fungal response to differ- medium (GMM) at pH 4.5 as described by Barad et al. ential gene regulation by pH; however, the overall response [7] and transferred 2 days later to buffered inducing remains poorly understood. media at pH 4 or 7. At several different time points after The diversity of recently described ecological interac- transfer to inducing secondary media (SM), fungal mycelia tions of Penicillium with fruits [3, 4] and with other were sampled and pooled for analysis. Pooled samples of Barad et al. BMC Genomics (2016) 17:330 Page 3 of 27

RNA were compared with independent samples 3 h after Group I comprised clusters of genes whose expression transfer to inducing media. patterns in the colonized tissue differed with respect to Principal component analysis (PCA) of all expressed growth at pH 4 or pH 7. This group comprised clusters genes of P. expansum indicated that the replicates were 1, 2, 3, and 6, which contained genes that are important very close to each other, and that the expression patterns for P. expansum colonization within apple fruits. of fungal genes exposed to the respective pH conditions Group II comprised clusters in which expression pat- for 3 h were almost identical to those of pooled samples terns of the colonized tissue were similar to those of the of mycelia exposed for various intervals (Fig. 1a). More- pathogen's growth in culture at pH 4, and differed from over, the general pattern of gene expression in decayed those of fungal growth at pH 7. This group includes apple tissue was far different from those of cultures grown clusters numbers 5, 7, and 8. under different pH conditions, as could be expected Group III comprised gene clusters in which expression (Fig. 1a). Hierarchical clustering analysis indicated differ- patterns of the colonized tissue were similar to those ing patterns of gene expression between the in vivo and in expressed by the pathogen grown in culture at pH 7. vitro samples at both pH levels; and the colonized apple This group includes clusters numbers 4 and 9 (Fig. 2). tissue contained more down- than up-regulated genes Group I includes clusters of expressed genes, whose (Fig. 1b). level of expression under in vitro conditions differed from Out of 11,019 annotated genes [12], 5,646 genes were that exhibited during colonization. This group comprised differentially expressed according to an false discovery clusters 1 and 2, with 2,248 and 233 genes, respectively, rate (FDR, [15]) threshold < 0.05 and the log fold change which exhibited higher expression under in vitro growth was greater than 1 or smaller than −1 implying at least 2 conditions at pH 4 or 7 than that manifested as down- fold change in expression scale (Fig. 1b). We sorted the regulation in the colonized apple tissue (Fig. 2). The genes differentially expressed gene patterns into nine different expressed under both high and low pH are probably im- co-expressed gene clusters, which fell into three major portant for growth under in vitro conditions but not for groups: pathogenicity. Group I also included clusters 3 and 6, with

Fig. 1 Principal component analysis (PCA) and heat map analysis of all the samples: duplicates of leading edge of inoculated apple; duplicates of pooled samples from all time points treated in vitro at pH 4.0; two replicates of P. expansum mycelia treated in vitro at pH 4.0 for 3 h; duplicates of a pool of samples from all time points treated in vitro at pH 7.0; and two replicates of P. expansum mycelia treated in vitro at pH 7.0 for 3 h. a PCA based on 5,646 differentially expressed genes. The results are plotted as: (♦) decayed apple; (■) in vitro at pH 4 for 3 h; (▲) pooled samples at pH 4; (X) in vitro at pH 7 for 3 h; (ӿ) pooled samples at pH 7. Circles are drawn around clustered points. b Expression heat map of differentially expressed P. expansum genes in the various samples Barad et al. BMC Genomics (2016) 17:330 Page 4 of 27

Fig. 2 Expression patterns of 9 clusters of co-expressed differential genes. The normalized expression pattern (log 2-transformed, median centered) of 9 clusters that were derived from the hierarchical clustering algorithm by use of the hclust function algorithm in R [70, 71]. Each gene is plotted in gray, in addition to the mean expression profile for that cluster (blue). The figure was generated by using perl script define_clusters_by_cutting_tree.pl of the Trinity software package [67]

2,220 and 72 genes, respectively, which comprise genes pH 7. These genes are probably necessary for growth that are up-regulated in apple tissue colonization but under alkaline conditions. down-regulated in vitro at both at pH 4 and pH 7. All the Group III comprises gene clusters that showed similar genes in this group probably are involved in growth in expression levels in colonized apple tissue and during in either nutritional environments or liquids, or both, that vitro growth fungal at pH 7, but showed altered gene ex- differ substantially from those within the apple tissue. pression at pH 4 (Fig. 2). This group included clusters 9 Group II comprised clusters 5, 7, and 8, in which the and 4, with 24 and 242 transcript counts, respectively. gene expression patterns in the colonized tissue were Genes in cluster 9 showed similar high expression levels in similar to those in vitro at pH 4 but different from those colonized fruit and in vitro at pH 7, but low levels in vitro in vitro at pH 7 (Fig. 2). Cluster 7, with a gene count of at pH 4; these genes probably are expressed in fruits under 67 transcripts, showed similar overall expression levels induced alkaline conditions or during fungal ammonia ac- during colonization of apple tissue and during in vitro cumulation [8]. Cluster 4 included genes that exhibited low growth at pH 4, which suggests that this cluster con- expression levels both in colonized fruit and in vitro at tained genes that are activated at pH 4, i.e., the usual pH 7; we hypothesize that these genes are repressed under fruit pH, therefore we consider that they may contribute alkaline conditions or during ammonia secretion [8]. to colonization at pH 4. Another two clusters present in this group were 5 and 8, with gene counts of 482 and Gene ontology analysis of decayed apple tissue 58, respectively; they include genes that were down- compared with in vitro conditions regulated both in colonized apple tissue and in vitro at We analyzed functional enrichment in each cluster using pH 4, but maintained high expression levels in vitro at blast2go software [16] and Fisher’s Exact Test [17]. Barad et al. BMC Genomics (2016) 17:330 Page 5 of 27

Clusters with different gene expression levels under genes encoding for aspartic endopeptidase-pep1-which is colonization than under in vitro conditions associated with pH modulation and pathogenicity of P. Higher expression levels of fungal genes when grown in digitatum [23]. Aspartic endopeptidase catalyzes hydroly- culture than in colonized tissue sis of elastin and collagen, the major structural proteins Genes in clusters 1 and 2 showed higher expression levels of basement membranes, and plays a significant role in under in vitro conditions than in colonized tissue. Func- virulence of P. digitatum on citrus fruits [23]. Aspartic tionality of these genes showed involvement in respiratory endopeptidase was up-regulated during infection of citrus activity, in light of their easier growth in culture media fruits, and contributed to fungal colonization, either by than in colonized apple tissue. GO-enriched terms in clus- degradation of plant cell-wall components to provide a ni- ters 1 and 2 are depicted in Additional file 1: Figure S1 trogen supply, or even by inactivating defense proteins and Additional file 2: Figure S2, respectively. [24]. This type of response at low pH is probably a result of accumulation of GLA during the pathogenicity process Lower expression levels of fungal genes when grown in of P. expansum, in order to ensure that secreted enzymes culture than in colonized tissue and metabolites are produced at the optimal pH to fully Genes in clusters 3 and 6 showed lower expression levels facilitate their physiological functions [3]. when grown in vitro at pH 4 or pH 7 than when grown in colonized tissue (Fig. 2). These clusters included genes Clusters with similar expression levels in colonized apple encoding for vesicle transport, which might indicate acti- and in vitro at pH 4 vation of secretion process(es); probably of mycotoxin(s) High expression level of fungal genes in colonized apple and effectors (Additional file 3: Figure S3 and Additional tissue and in samples grown in culture at pH 4 file 4: Figure S6 respectively). Cluster 3 included most of A single cluster-cluster 7-showed enhanced expression of the 15 genes involved in patulin biosynthesis-patB, D, E, F, genes both in colonized apple tissue and in vitro at pH 4 H, I, J, K, L, and O. Unexpectedly, these genes were not (Fig. 2). This cluster was enriched in genes involved in the highly expressed in vitro at either pH 4 or pH 7, which glutamate metabolic process (Additional file 5: Figure S7); supports a previous report by Li et al. [18], which sug- a process that includes high expression of glutamate gested the importance of specific nutritional growth con- decarboxylase (Table 1), which is required for normal ditions for induced expression of the patulin biosynthesis oxidative-stress tolerance in Saccharomyces cerevisiae. gene cluster (PatA-PatO) in P. expansum.However,at γ-Aminobutyric acid (GABA) largely originates from the same time, the question arises of the specific mech- decarboxylation of L-glutamate; it is associated with anism(s)-possibly nutritional and possibly others-that sporulation/spore metabolism [25] and in most fungi modulate the conditions for expression of the mycotoxin it serves as a carbon and nitrogen source. Also, γ- cluster in vivo [18–20]. Cluster 3 also showed genes such aminobutyric acid metabolism promotes asexual sporula- as acetamidase, amidase-family proteins, and fatty-acid tion in Saccharomyces nodorum [26]. Moreover, in plants amide hydrolase, encoding for amidase activity (Additional and fungi GABA synthesis has been associated with acidic file 3: Figure S3) these may indicate a possible source of pH, either in response to cytosolic acidification-probably + NH4 production during periods of limited sucrose levels as a pH-regulatory mechanism-or during growth under and pathogenicity of P. expansum, which would support acidic conditions [25]. Since acidification of the host envir- earlier suggestions by Barad et al. [8] that local pH modu- onment usually occurs during apple colonization by P. lation might result from ammonia accumulation in the expansum, it is possible that γ-aminobutyric acid synthesis leading edge during pathogenicity of P. expansum. might be one of the mechanisms employed by the fungus Cluster 6 shows enriched expression of genes associated to cope with this more acidic pH of the environment [6]. with host-cell-wall degradation that are important for the virulence of P. expansum [5] (Additional file 4: Figure. S6). Low expression level of fungal genes in colonized apple and These genes have previously known functionalities associ- in samples grown in vitro at pH 4 ated with pathogenicity of fungi; these genes and activities In contrast to the single up-regulated cluster 7, two include: chitinase-associated genes, pectin lyase, and poly- clusters-5 and 8-showed repressed expression of genes galacturonase (PG) activities. Among these, PG was shown both in colonized apple tissue and in vitro at pH 4 to be involved in maceration of apples by P. expansum, (Fig. 2). Cluster 5 showed enrichment of genes that are particularly in diseases characterized by tissue maceration involved in fungal pathogenicity and that are known to or soft rot [21]; and pectin lyases contribute to the patho- be active under alkaline conditions; they exhibit, for genicity of many pathogens by degrading pectin polymers example, pectate lyase and hydrolase activities [27] directly by means of a β-elimination mechanism that re- (Additional file 6: Figure S5) and the transcription factor sults in formation of 4,5-unsaturated oligo-galacturonides PacC (Table 2), which suggests that the acidic pH-repressed [22]. In addition, cluster 6 shows over-representation of alkaline-expressed genes are involved in pathogenicity. It is Barad et al. BMC Genomics (2016) 17:330 Page 6 of 27

Table 1 Genes involved in each process of cluster 7 GO Term Gene id Genbank Description transcript id Nucleobase transport PEXP_081890 KGO47390 Permease, cytosine/purine, uracil, thiamine, allantoin PEXP_004400 KGO37467 Permease, cytosine/purine, uracil, thiamine, allantoin PEXP_081870 KGO47388 Permease, cytosine/purine, uracil, thiamine, allantoin Taurine metabolic process PEXP_099510 KGO46394 Pyridoxal phosphate-dependent transferase, major region, subdomain 1 PEXP_039450 KGO48081 Pyridoxal phosphate-dependent transferase, major region, subdomain 1 Beta-alanine metabolic process PEXP_099510 KGO46394 Pyridoxal phosphate-dependent transferase, major region, subdomain 1 PEXP_039450 KGO48081 Pyridoxal phosphate-dependent transferase, major region, subdomain 1 Transmembrane transport PEXP_023020 KGO42773 Major facilitator superfamily domain, general substrate transporter PEXP_104460 KGO36460 Major facilitator superfamily domain, general substrate transporter PEXP_019050 KGO45782 C4-dicarboxylate transporter/malic acid transport protein PEXP_083120 KGO38619 Major facilitator superfamily domain, general substrate transporter PEXP_105230 KGO41447 Major facilitator superfamily domain, general substrate transporter PEXP_012370 KGO49188 Major facilitator superfamily domain, general substrate transporter PEXP_060340 KGO45245 Iron permease FTR1 PEXP_059590 KGO45345 Major facilitator superfamily domain, general substrate transporter PEXP_094700 KGO43568 Major facilitator superfamily domain, general substrate transporter PEXP_025230 KGO42652 Major facilitator superfamily domain, general substrate transporter PEXP_030850 KGO40270 ABC transporter, integral membrane type 1 PEXP_078840 KGO37868 Major facilitator superfamily domain, general substrate transporter Glutamate metabolic process PEXP_099510 KGO46394 Pyridoxal phosphate-dependent transferase, major region, subdomain 1 PEXP_039450 KGO48081 Pyridoxal phosphate-dependent transferase, major region, subdomain 1 Glutamate decarboxylase activity PEXP_099510 KGO46394 Pyridoxal phosphate-dependent transferase, major region, subdomain 1 PEXP_039450 KGO48081 Pyridoxal phosphate-dependent transferase, major region, subdomain 1 Nucleobase transmembrane transporter PEXP_081890 KGO47390 Permease, cytosine/purine, uracil, thiamine, allantoin activity PEXP_004400 KGO37467 Permease, cytosine/purine, uracil, thiamine, allantoin PEXP_081870 KGO47388 Permease, cytosine/purine, uracil, thiamine, allantoin Cofactor binding PEXP_107000 KGO41320 Aldolase-type TIM barrel PEXP_068570 KGO46687 Aldolase-type TIM barrel PEXP_099510 KGO46394 Pyridoxal phosphate-dependent transferase, major region, subdomain 1 PEXP_080260 KGO38010 Pyridoxal phosphate-dependent transferase, major region, subdomain 2 PEXP_043260 KGO39485 Thiamine pyrophosphate enzyme, C-terminal TPP-binding PEXP_076130 KGO36094 D-isomer specific 2-hydroxyacid dehydrogenase, NAD-binding PEXP_001130 KGO44422 Glyceraldehyde/Erythrose phosphate dehydrogenase family PEXP_039450 KGO48081 Pyridoxal phosphate-dependent transferase, major region, subdomain 1 Glucosylceramidase activity PEXP_081260 KGO47327 Glycoside hydrolase, family 30 3-isopropylmalate dehydratase activity PEXP_074470 KGO48717 Aconitase/3-isopropylmalate dehydratase large subunit, alpha/beta/ alpha, subdomain 1/3 Barad et al. BMC Genomics (2016) 17:330 Page 7 of 27

Table 1 Genes involved in each process of cluster 7 (Continued) N-acylphosphatidylethanolamine-specific PEXP_024950 KGO42624 N-acyl-phosphatidylethanolamine-hydrolysing phospholipase D phospholipase D activity Transmembrane transporter activity PEXP_104460 KGO36460 Major facilitator superfamily domain, general substrate transporter PEXP_034210 KGO40038 CDR ABC transporter PEXP_019050 KGO45782 C4-dicarboxylate transporter/malic acid transport protein PEXP_105230 KGO41447 Major facilitator superfamily domain, general substrate transporter PEXP_081890 KGO47390 Permease, cytosine/purine, uracil, thiamine, allantoin PEXP_004400 KGO37467 Permease, cytosine/purine, uracil, thiamine, allantoin PEXP_030850 KGO40270 ABC transporter, integral membrane type 1 PEXP_081870 KGO47388 Permease, cytosine/purine, uracil, thiamine, allantoin Integral component of membrane PEXP_023020 KGO42773 Major facilitator superfamily domain, general substrate transporter PEXP_104460 KGO36460 Major facilitator superfamily domain, general substrate transporter PEXP_034210 KGO40038 CDR ABC transporter PEXP_019050 KGO45782 C4-dicarboxylate transporter/malic acid transport protein PEXP_083120 KGO38619 Major facilitator superfamily domain, general substrate transporter PEXP_054930 KGO44009 Major intrinsic protein PEXP_105230 KGO41447 Major facilitator superfamily domain, general substrate transporter PEXP_012370 KGO49188 Major facilitator superfamily domain, general substrate transporter PEXP_059590 KGO45345 Major facilitator superfamily domain, general substrate transporter PEXP_094700 KGO43568 Major facilitator superfamily domain, general substrate transporter PEXP_027630 KGO43014 Amino acid transporter, transmembrane PEXP_025230 KGO42652 Major facilitator superfamily domain, general substrate transporter PEXP_030850 KGO40270 ABC transporter, integral membrane type 1 PEXP_038200 KGO47945 Mitochondrial substrate/solute carrier PEXP_078840 KGO37868 Major facilitator superfamily domain, general substrate transporter Lysosome PEXP_081260 KGO47327 Glycoside hydrolase, family 30 3-Isopropylmalate dehydratase complex PEXP_074470 KGO48717 Aconitase/3-isopropylmalate dehydratase large subunit, alpha/beta/ alpha, subdomain 1/3 likely that P. expansum adopted its acidifying life pattern [8]. This behavior may indicate that these processes are characterized by production of organic acids-mainly usually repressed during colonization unless there is a sig- gluconic acid secretion [7]-in order to optimize the pH nificant increase in ammonia accumulation. of the media. These present findings support a previous publication by Barad et al. [28] which indicated that Clusters with similar expression levels in colonized apple whereas pacC expression was inhibited under acidic tissue and in vitro at pH 7 conditions during the accumulation of gluconic acid, it High expression level of fungal genes in colonized apple might be activated in the leading edge of colonized tis- tissue and in fungi grown in vitro at pH 7 sue where ammonia is accumulated at pH 4 [8]. Part of One of the most important clusters that showed high the localized pH modulation may be regulated by the expression levels both in colonized apple tissue and in Na+ and Ca2+ transporters that are present in cluster 5, vitro at pH 7 was number 9 (Fig. 2), which showed sig- that modulate the internal equilibrium of hydrogen nificant enrichment of cellular amino-acid metabolism ions and contribute to cytoplasmic pH (Table 2). (Table 4, Additional file 8: Figure S9). Ammonia accumu- A second cluster that was under-represented, both in lation is induced under the limited-nutrient conditions colonized apple tissue and in vitro at pH 4 was cluster 8 present at the leading edge of the decay, in order to enable (Fig. 2). This cluster mainly showed enrichment in the pathogen to use a battery of pectolytic enzymes for tis- carbon utilization and sugar-transport-related genes sue maceration [8]. Among the genes involved in cellular (Table 3, Additional file 7: Figure S8), which suggests amino-acid metabolism process is glucose-methanol- that they contribute to a series of processes that induce car- choline oxidoreductase (GMC) (Table 4), whose families bon catabolism and thereby result in GLA accumulation are clusters of FAD flavoprotein oxidoreductases-highly Barad et al. BMC Genomics (2016) 17:330 Page 8 of 27

Table 2 Genes involved in each process of cluster 5 GO Term Gene id Genbank Description transcript id Regulation of pH PEXP_046990 KGO39741 Sodium/calcium exchanger membrane region PEXP_043720 KGO39291 Alkali metal cation/H+ antiporter Nha1, C-terminal PEXP_009670 KGO49300 Cation/H+ exchanger PEXP_095400 KGO43638 Sodium/calcium exchanger membrane region L-Arabinose metabolic process PEXP_023030 KGO42774 Glycoside hydrolase, superfamily PEXP_071150 KGO40611 Alcohol dehydrogenase superfamily, zinc-type PEXP_089320 KGO41720 Glycoside hydrolase, superfamily Calcium ion transport PEXP_046990 KGO39741 Sodium/calcium exchanger membrane region PEXP_088420 KGO41618 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter PEXP_095400 KGO43638 Sodium/calcium exchanger membrane region PEXP_050270 KGO42077 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter Kynurenine metabolic process PEXP_039010 KGO48037 putative cyclase PEXP_045070 KGO39237 putative cyclase PEXP_009350 KGO49268 Pyridoxal phosphate-dependent transferase, major region, subdomain 2 Glyoxylate cycle PEXP_011610 KGO49112 Malate synthase A PEXP_077890 KGO37773 Pyruvate/Phosphoenolpyruvate kinase Tryptophan catabolic process PEXP_039010 KGO48037 Putative cyclase PEXP_045070 KGO39237 Putative cyclase PEXP_009350 KGO49268 Pyridoxal phosphate-dependent transferase, major region, subdomain 2 Inorganic ion transmembrane transport PEXP_059490 KGO45335 Sulfate anion transporter PEXP_043720 KGO39291 Alkali metal cation/H+ antiporter Nha1, C-terminal PEXP_048620 KGO39686 Nickel/cobalt transporter, high-affinity PEXP_009670 KGO49300 Cation/H+ exchanger PEXP_088420 KGO41618 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter PEXP_095400 KGO43638 Sodium/calcium exchanger membrane region PEXP_050270 KGO42077 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter PEXP_094650 KGO43563 Ammonium transporter Disaccharide metabolic process PEXP_052570 KGO42305 Glycosyl transferase, family 20 PEXP_011870 KGO49138 Pectinesterase, catalytic PEXP_069330 KGO46763 Glycoside hydrolase, family 61 PEXP_011880 KGO49139 Glycoside hydrolase, family 28 PEXP_066270 KGO46936 Hexokinase, N-terminal PEXP_053240 KGO42372 Glycoside hydrolase, family 61 PEXP_107940 KGO41788 Alpha-amylase, C-terminal all beta Substrate-specific transmembrane transporter PEXP_046990 KGO39741 Sodium/calcium exchanger membrane region activity PEXP_059490 KGO45335 Sulfate anion transporter PEXP_016910 KGO46049 Xanthine/uracil/vitamin C permease PEXP_043720 KGO39291 Alkali metal cation/H+ antiporter Nha1, C-terminal PEXP_052330 KGO42281 Major facilitator superfamily domain, general substrate transporter PEXP_033600 KGO39977 Amino acid/polyamine transporter I PEXP_094860 KGO43584 Major facilitator superfamily domain, general substrate transporter PEXP_068270 KGO46657 Nucleobase cation symporter-1, NCS1 Barad et al. BMC Genomics (2016) 17:330 Page 9 of 27

Table 2 Genes involved in each process of cluster 5 (Continued) PEXP_006100 KGO36811 Major facilitator superfamily domain, general substrate transporter PEXP_086200 KGO40808 Amino acid/polyamine transporter I PEXP_003610 KGO37353 Major facilitator superfamily domain, general substrate transporter PEXP_043410 KGO39260 Major facilitator superfamily domain, general substrate transporter PEXP_048620 KGO39686 Nickel/cobalt transporter, high-affinity PEXP_006360 KGO36837 Major facilitator superfamily domain, general substrate transporter PEXP_000260 KGO44335 Amino acid/polyamine transporter I PEXP_072400 KGO48510 Major facilitator superfamily domain, general substrate transporter PEXP_009030 KGO48810 Na dependent nucleoside transporter PEXP_068910 KGO46721 Major facilitator superfamily domain, general substrate transporter PEXP_031270 KGO40312 Permease, cytosine/purine, uracil, thiamine, allantoin PEXP_009670 KGO49300 Cation/H+ exchanger PEXP_110420 KGO38764 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter PEXP_095790 KGO43677 Major facilitator superfamily domain, general substrate transporter PEXP_048590 KGO39683 Amino acid/polyamine transporter I PEXP_104220 KGO36561 Cation/H+ exchanger PEXP_019490 KGO45826 C4-dicarboxylate transporter/malic acid transport protein PEXP_088420 KGO41618 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter PEXP_076320 KGO37552 Major facilitator superfamily domain, general substrate transporter PEXP_001370 KGO37112 Major facilitator superfamily domain, general substrate transporter PEXP_084160 KGO41053 Amino acid/polyamine transporter I PEXP_095400 KGO43638 Sodium/calcium exchanger membrane region PEXP_050270 KGO42077 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter PEXP_062420 KGO45644 General substrate transporter PEXP_022750 KGO42746 Major facilitator superfamily domain, general substrate transporter PEXP_068690 KGO46699 Major facilitator superfamily domain, general substrate transporter PEXP_034320 KGO40048 Arsenical pump membrane protein, ArsB PEXP_074940 KGO36159 Major facilitator superfamily domain, general substrate transporter PEXP_094650 KGO43563 Ammonium transporter Solute:proton antiporter activity PEXP_046990 KGO39741 Sodium/calcium exchanger membrane region PEXP_043720 KGO39291 Alkali metal cation/H+ antiporter Nha1, C-terminal PEXP_009670 KGO49300 Cation/H+ exchanger PEXP_104220 KGO36561 Cation/H+ exchanger PEXP_095400 KGO43638 Sodium/calcium exchanger membrane region Cation:cation antiporter activity PEXP_046990 KGO39741 Sodium/calcium exchanger membrane region PEXP_043720 KGO39291 Alkali metal cation/H+ antiporter Nha1, C-terminal PEXP_104220 KGO36561 Cation/H+ exchanger PEXP_095400 KGO43638 Sodium/calcium exchanger membrane region Metal ion binding PEXP_046510 KGO39177 Cytochrome P450, E-class, CYP52 PEXP_036890 KGO35901 Transcription factor, fungi PEXP_084560 KGO41093 4-Hydroxyphenylpyruvate dioxygenase PEXP_055320 KGO44077 Polyketide synthase, enoylreductase PEXP_042410 KGO39400 Manganese/iron superoxide dismutase, C-terminal PEXP_072700 KGO48540 Annexin PEXP_089160 KGO41704 Terpenoid synthase Barad et al. BMC Genomics (2016) 17:330 Page 10 of 27

Table 2 Genes involved in each process of cluster 5 (Continued) PEXP_102680 KGO36423 Transcription factor, fungi PEXP_042010 KGO48399 Protein of unknown function DUF3468 PEXP_011320 KGO49083 Zinc finger, C2H2 PEXP_095680 KGO43666 Protein of unknown function DUF3468 PEXP_050580 KGO42108 Cytochrome P450 PEXP_059140 KGO45293 Polyketide synthase, enoylreductase PEXP_100050 KGO46448 Heavy metal-associated domain, HMA PEXP_103630 KGO36296 Urease accessory protein UreD PEXP_083550 KGO40992 Thiamine pyrophosphate enzyme, C-terminal TPP-binding PEXP_070880 KGO40584 Polyketide synthase, enoylreductase PEXP_027120 KGO42963 hypothetical protein PEXP_040610 KGO48229 Transcription factor, fungi PEXP_075350 KGO36016 Alpha-actinin PEXP_011040 KGO49055 Cytochrome P450, E-class, group I PEXP_005060 KGO36707 Transcription factor, fungi PEXP_070870 KGO40583 Aldehyde dehydrogenase, C-terminal PEXP_046450 KGO39171 Amidohydrolase 1 PEXP_008360 KGO37036 4-Hydroxyphenylpyruvate dioxygenase PEXP_048620 KGO39686 Nickel/cobalt transporter, high-affinity PEXP_101160 KGO38158 Pectin lyase fold/virulence factor PEXP_022330 KGO42704 Transcription factor, fungi PEXP_058230 KGO38945 Alcohol dehydrogenase superfamily, zinc-type PEXP_053390 KGO42387 Polyketide synthase, enoylreductase PEXP_017020 KGO46060 Cytochrome P450 PEXP_021500 KGO44639 Transcription factor, fungi PEXP_043440 KGO39263 Polyketide synthase, enoylreductase PEXP_102420 KGO36222 Protein of unknown function DUF3468 PEXP_108970 KGO41891 Polyketide synthase, enoylreductase PEXP_095570 KGO43655 Transcription factor, fungi PEXP_088350 KGO41611 Transcription factor, fungi PEXP_056220 KGO38423 Cytochrome P450 PEXP_099120 KGO46300 Hypothetical protein PEXP_028450 KGO43127 Transcription factor, fungi PEXP_110420 KGO38764 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter PEXP_050400 KGO42090 Zinc finger, C2H2 PEXP_088420 KGO41618 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter PEXP_085230 KGO41160 Hypothetical protein PEXP_000560 KGO44365 Class II aldolase/adducin N-terminal PEXP_071150 KGO40611 Alcohol dehydrogenase superfamily, zinc-type PEXP_090470 KGO45017 Pyruvate carboxyltransferase PEXP_080790 KGO38063 hypothetical protein PEXP_026670 KGO42918 Transcription factor, fungi PEXP_084100 KGO41047 Transcription factor, fungi PEXP_102160 KGO38273 Casein kinase II, regulatory subunit PEXP_036970 KGO35909 Cytochrome P450, E-class, group I Barad et al. BMC Genomics (2016) 17:330 Page 11 of 27

Table 2 Genes involved in each process of cluster 5 (Continued) PEXP_032030 KGO39788 Hypothetical protein PEXP_089350 KGO41723 Transcription factor, fungi PEXP_005230 KGO36724 Transcription factor, fungi PEXP_057180 KGO44225 ATP adenylyltransferase, C-terminal PEXP_002560 KGO37249 Hypothetical protein PEXP_081210 KGO47322 ATP-grasp fold, subdomain 1 PEXP_071070 KGO40603 Molybdenum cofactor synthesis C-terminal PEXP_095390 KGO43637 Transcription factor, fungi PEXP_054870 KGO44003 Transcription factor, fungi PEXP_042700 KGO39429 Hypothetical protein PEXP_036990 KGO35911 Ureohydrolase PEXP_041200 KGO48300 Transcription factor, fungi PEXP_050270 KGO42077 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter PEXP_048130 KGO39637 Transcription factor, fungi PEXP_010010 KGO48952 Transcription factor, fungi PEXP_085290 KGO41166 Transcription factor, fungi PEXP_079380 KGO37922 Zinc finger, C2H2 PEXP_057190 KGO44226 Thiamine pyrophosphate enzyme, C-terminal TPP-binding PEXP_069040 KGO46734 Cytochrome P450 PEXP_007560 KGO36956 Zinc finger, C2H2 PEXP_081220 KGO47323 Transcription factor, fungi PEXP_087170 KGO40934 Transcription factor, fungi PEXP_105830 KGO41509 Transcription factor, fungi PEXP_043140 KGO39473 Cytochrome P450 PEXP_067370 KGO46568 hypothetical protein PEXP_073070 KGO48577 Exonuclease, RNase T/DNA polymerase III PEXP_051020 KGO42152 Amidohydrolase 1 PEXP_061400 KGO45512 Hypothetical protein PEXP_101010 KGO38143 Glycoside hydrolase, family 71 PEXP_043150 KGO39474 Terpenoid synthase PEXP_015320 KGO47645 ATP-grasp fold, subdomain 1 Pectate lyase activity PEXP_101160 KGO38158 Pectin lyase fold/virulence factor PEXP_080220 KGO38006 Pectate lyase, catalytic Calcium ion transmembrane transporter activity PEXP_046990 KGO39741 Sodium/calcium exchanger membrane region PEXP_088420 KGO41618 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter PEXP_095400 KGO43638 Sodium/calcium exchanger membrane region PEXP_050270 KGO42077 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter Sequence-specific DNA binding RNA polymerase PEXP_102680 KGO36423 Transcription factor, fungi II transcription factor activity PEXP_042010 KGO48399 Protein of unknown function DUF3468 PEXP_095680 KGO43666 Protein of unknown function DUF3468 PEXP_040610 KGO48229 Transcription factor, fungi PEXP_070870 KGO40583 Aldehyde dehydrogenase, C-terminal PEXP_021500 KGO44639 Transcription factor, fungi PEXP_102420 KGO36222 Protein of unknown function DUF3468 PEXP_088350 KGO41611 Transcription factor, fungi Barad et al. BMC Genomics (2016) 17:330 Page 12 of 27

Table 2 Genes involved in each process of cluster 5 (Continued) PEXP_099120 KGO46300 Hypothetical protein PEXP_085230 KGO41160 Hypothetical protein PEXP_080790 KGO38063 Hypothetical protein PEXP_102160 KGO38273 Casein kinase II, regulatory subunit PEXP_032030 KGO39788 Hypothetical protein PEXP_005230 KGO36724 Transcription factor, fungi PEXP_057180 KGO44225 ATP adenylyltransferase, C-terminal PEXP_002560 KGO37249 Hypothetical protein PEXP_095390 KGO43637 Transcription factor, fungi PEXP_042700 KGO39429 Hypothetical protein PEXP_048130 KGO39637 Transcription factor, fungi PEXP_010010 KGO48952 Transcription factor, fungi PEXP_085290 KGO41166 Transcription factor, fungi PEXP_087170 KGO40934 Transcription factor, fungi PEXP_067370 KGO46568 Hypothetical protein PEXP_101010 KGO38143 Glycoside hydrolase, family 71 Hydrolase activity, hydrolyzing O-glycosyl PEXP_000940 KGO44403 Protein of unknown function DUF2985 compounds PEXP_044190 KGO39338 Aldolase-type TIM barrel PEXP_049330 KGO39518 Glycoside hydrolase, family 35 PEXP_023030 KGO42774 Glycoside hydrolase, superfamily PEXP_101680 KGO38226 Concanavalin A-like lectin/glucanases superfamily PEXP_023340 KGO42463 Glycoside hydrolase, family 43 PEXP_070780 KGO40574 Glycoside hydrolase, family 31 PEXP_072880 KGO48558 Glycoside hydrolase, superfamily PEXP_048880 KGO39711 Concanavalin A-like lectin/glucanases superfamily PEXP_069330 KGO46763 Glycoside hydrolase, family 61 PEXP_011880 KGO49139 Glycoside hydrolase, family 28 PEXP_089320 KGO41720 Glycoside hydrolase, superfamily PEXP_076310 KGO37551 Glycoside hydrolase, family 32 PEXP_026640 KGO42915 Glycoside hydrolase, family 16, CRH1, predicted PEXP_057570 KGO44264 Glycoside hydrolase family 3 PEXP_053240 KGO42372 Glycoside hydrolase, family 61 PEXP_042140 KGO48412 Concanavalin A-like lectin/glucanase, subgroup PEXP_107940 KGO41788 Alpha-amylase, C-terminal all beta ATPase activity, coupled to transmembrane PEXP_023060 KGO42777 CDR ABC transporter movement of substances PEXP_050880 KGO42138 ABC transporter, integral membrane type 1 PEXP_110420 KGO38764 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter PEXP_040720 KGO48240 ABC transporter, integral membrane type 1 PEXP_071990 KGO40700 ABC transporter, integral membrane type 1 PEXP_088420 KGO41618 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter PEXP_104880 KGO41412 ABC transporter, integral membrane type 1 PEXP_050270 KGO42077 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter PEXP_077310 KGO37715 CDR ABC transporter PEXP_086490 KGO40844 ABC transporter, integral membrane type 1 PEXP_057530 KGO44260 ABC transporter, integral membrane type 1 Barad et al. BMC Genomics (2016) 17:330 Page 13 of 27

Table 2 Genes involved in each process of cluster 5 (Continued) 4-Hydroxyphenylpyruvate dioxygenase activity PEXP_084560 KGO41093 4-Hydroxyphenylpyruvate dioxygenase PEXP_008360 KGO37036 4-Hydroxyphenylpyruvate dioxygenase Integral component of membrane PEXP_046990 KGO39741 Sodium/calcium exchanger membrane region PEXP_024890 KGO42618 Major facilitator superfamily domain, general substrate transporter PEXP_041670 KGO48366 Major facilitator superfamily domain, general substrate transporter PEXP_059490 KGO45335 Sulfate anion transporter PEXP_043720 KGO39291 Alkali metal cation/H+ antiporter Nha1, C-terminal PEXP_052330 KGO42281 Major facilitator superfamily domain, general substrate transporter PEXP_037770 KGO36126 Major facilitator superfamily domain, general substrate transporter PEXP_102860 KGO36441 Mitochondrial carrier protein PEXP_023060 KGO42777 CDR ABC transporter PEXP_033600 KGO39977 Amino acid/polyamine transporter I PEXP_014370 KGO47550 Amino acid transporter, transmembrane PEXP_094860 KGO43584 Major facilitator superfamily domain, general substrate transporter PEXP_006100 KGO36811 Major facilitator superfamily domain, general substrate transporter PEXP_046430 KGO39169 Major facilitator superfamily domain, general substrate transporter PEXP_086200 KGO40808 Amino acid/polyamine transporter I PEXP_024440 KGO42573 Major facilitator superfamily domain, general substrate transporter PEXP_003610 KGO37353 Major facilitator superfamily domain, general substrate transporter PEXP_043410 KGO39260 Major facilitator superfamily domain, general substrate transporter PEXP_048620 KGO39686 Nickel/cobalt transporter, high-affinity PEXP_006360 KGO36837 Major facilitator superfamily domain, general substrate transporter PEXP_044620 KGO39381 Tetracycline resistance protein, TetA/multidrug resistance protein MdtG PEXP_072400 KGO48510 Major facilitator superfamily domain, general substrate transporter PEXP_028370 KGO43119 Amino acid transporter, transmembrane PEXP_009920 KGO48943 Peroxisomal biogenesis factor 11 PEXP_033410 KGO39958 Major facilitator superfamily domain, general substrate transporter PEXP_050880 KGO42138 ABC transporter, integral membrane type 1 PEXP_056980 KGO44205 Major facilitator superfamily domain, general substrate transporter PEXP_102420 KGO36222 Protein of unknown function DUF3468 PEXP_074410 KGO48711 Major facilitator superfamily domain, general substrate transporter PEXP_068910 KGO46721 Major facilitator superfamily domain, general substrate transporter PEXP_060610 KGO45433 Major facilitator superfamily domain, general substrate transporter PEXP_009670 KGO49300 Cation/H+ exchanger PEXP_017290 KGO46087 Major facilitator superfamily domain, general substrate transporter PEXP_110420 KGO38764 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter PEXP_059360 KGO45322 Mitochondrial carrier protein PEXP_095790 KGO43677 Major facilitator superfamily domain, general substrate transporter PEXP_040720 KGO48240 ABC transporter, integral membrane type 1 PEXP_071990 KGO40700 ABC transporter, integral membrane type 1 PEXP_048590 KGO39683 Amino acid/polyamine transporter I PEXP_104220 KGO36561 Cation/H+ exchanger PEXP_000120 KGO44321 Major facilitator superfamily domain, general substrate transporter PEXP_019490 KGO45826 C4-dicarboxylate transporter/malic acid transport protein PEXP_088420 KGO41618 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter Barad et al. BMC Genomics (2016) 17:330 Page 14 of 27

Table 2 Genes involved in each process of cluster 5 (Continued) PEXP_076320 KGO37552 Major facilitator superfamily domain, general substrate transporter PEXP_001370 KGO37112 Major facilitator superfamily domain, general substrate transporter PEXP_001360 KGO37111 Major facilitator superfamily domain, general substrate transporter PEXP_104880 KGO41412 ABC transporter, integral membrane type 1 PEXP_005240 KGO36725 Major facilitator superfamily domain, general substrate transporter PEXP_108540 KGO41848 Major facilitator superfamily domain, general substrate transporter PEXP_095400 KGO43638 Sodium/calcium exchanger membrane region PEXP_046610 KGO39187 Sodium/calcium exchanger membrane region PEXP_098820 KGO46270 Major facilitator superfamily domain, general substrate transporter PEXP_001950 KGO37170 Mitochondrial carrier protein PEXP_050270 KGO42077 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter PEXP_023180 KGO42789 Major facilitator superfamily domain, general substrate transporter PEXP_010400 KGO48991 Major facilitator superfamily domain, general substrate transporter PEXP_098540 KGO46242 Major facilitator superfamily domain, general substrate transporter PEXP_066250 KGO46934 Major facilitator superfamily domain, general substrate transporter PEXP_062420 KGO45644 General substrate transporter PEXP_026170 KGO42868 Major facilitator superfamily domain, general substrate transporter PEXP_077310 KGO37715 CDR ABC transporter PEXP_022750 KGO42746 Major facilitator superfamily domain, general substrate transporter PEXP_068690 KGO46699 Major facilitator superfamily domain, general substrate transporter PEXP_066860 KGO46517 Major facilitator superfamily domain, general substrate transporter PEXP_086490 KGO40844 ABC transporter, integral membrane type 1 PEXP_067360 KGO46567 Major facilitator superfamily domain, general substrate transporter PEXP_057530 KGO44260 ABC transporter, integral membrane type 1 PEXP_034320 KGO40048 Arsenical pump membrane protein, ArsB PEXP_074940 KGO36159 Major facilitator superfamily domain, general substrate transporter complex genes that contribute to several oxidation and re- Heat-map analysis of this family showed that genes with duction processes [29]. These enzymes include a variety of the same activity were differentially expressed between dif- proteins, not all of which were present in cluster 9, such ferent clusters (Fig. 4). For example, 11 GMC oxidoreduc- as choline dehydrogenase (CHD), which was present in tase genes were detected in P. expansum: three in cluster clusters 1, 3, and 9, methanol oxidase (MOX), and cellobi- 1, three in cluster 3, and one in cluster 9, whereas the ose dehydrogenase, which was present in cluster 3; these remaining four were not differentially expressed. The are proteins that share a number of homologous regions GMC oxidoreductase family also exhibited close similarity that show sequence similarities. Since ammonia accumula- to the glucose oxidase family. We were able to detect three tion at the leading edge of the Penicillium-colonized tissue glucose oxidase transcripts, as described by Ballester et al. did not increase local pH [8], it is possible that the accu- [12], which showed differing expression patterns: gox1 of mulated ammonia may activate gene expression, either P. expansum was not differentially expressed under our directly or indirectly by generation of hydrogen peroxide conditions, gox2 was found in cluster 3, and gox3 was de- and activation of RBOH in the killed cell [30]. To elucidate tected in cluster 1 (Fig. 4). Analysis of only the expression the role of ammonia, we analyzed the relative expression pattern at pH 4 compared with that at pH 7 showed that levels of several genes-MepB, CuAC and ACC-from clus- both gox2 and glucose oxidase 3 (gox3) were upregulated ter 9 in Penicillium-colonized fruits exposed to 22 μMof at pH 7 but not at pH 4 [7]. This differential expression + NH4, and found induction of their relative expression pattern of the gox family suggests that genes were being levels (Fig. 3), which indicates the capability of ammonia selectively activated on the basis of their optimal condi- to activate this process. tions with respect to pH, in vitro, or in vivo, to enable the Another highly expressed family of genes in cluster 9 fungus to cope with varied conditions and to make opti- was the glucose-methanol-choline oxidoreductase family. mal use of the inventory of available enzymes [31]. Barad et al. BMC Genomics (2016) 17:330 Page 15 of 27

Table 3 Genes involved in each process of cluster 8 GO Term Gene id Genbank transcript id Description Phosphate ion transport PEXP_088430 KGO41619 Phosphate transporter PEXP_030290 KGO40453 Phosphate transporter Carbon utilization PEXP_105770 KGO41503 Ribose/galactose isomerase PEXP_105760 KGO41502 Aldolase-type TIM barrel Transmembrane transport PEXP_104850 KGO41409 Major facilitator superfamily domain, general substrate transporter PEXP_048020 KGO39626 Major facilitator superfamily domain, general substrate transporter PEXP_070650 KGO40561 Major facilitator superfamily domain, general substrate transporter PEXP_011530 KGO49104 Major facilitator superfamily domain, general substrate transporter PEXP_033090 KGO39926 Amino acid/polyamine transporter I PEXP_078910 KGO37875 Cation efflux protein PEXP_049390 KGO39524 Cation/H+ exchanger PEXP_030290 KGO40453 Phosphate transporter PEXP_034270 KGO40044 Major facilitator superfamily domain, general substrate transporter Cation transport PEXP_002160 KGO37191 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter PEXP_078910 KGO37875 Cation efflux protein PEXP_049390 KGO39524 Cation/H+ exchanger PEXP_004260 KGO37441 Sodium\x3aneurotransmitter symporter Neurotransmitter transport PEXP_004260 KGO37441 Sodium\x3aneurotransmitter symporter Triose-phosphate isomerase activity PEXP_105760 KGO41502 Aldolase-type TIM barrel Ribose-5-phosphate isomerase activity PEXP_105770 KGO41503 Ribose/galactose isomerase Cysteine dioxygenase activity PEXP_086730 KGO40868 Cysteine dioxygenase type I asparaginase activity PEXP_042000 KGO48398 L-asparaginase, type II Integral component of membrane PEXP_002160 KGO37191 ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter PEXP_104850 KGO41409 Major facilitator superfamily domain, general substrate transporter PEXP_025010 KGO42630 Hypothetical protein PEXP_083360 KGO38643 FAD-binding 8 PEXP_048020 KGO39626 Major facilitator superfamily domain, general substrate transporter PEXP_070650 KGO40561 Major facilitator superfamily domain, general substrate transporter PEXP_011530 KGO49104 Major facilitator superfamily domain, general substrate transporter PEXP_033090 KGO39926 Amino acid/polyamine transporter I PEXP_078910 KGO37875 Cation efflux protein PEXP_049390 KGO39524 Cation/H+ exchanger PEXP_004260 KGO37441 Sodium\x3aneurotransmitter symporter PEXP_034270 KGO40044 Major facilitator superfamily domain, general substrate transporter

Another process that was enriched in this cluster was at their primary amino groups [33]. The products of putres- amine metabolism (Additional file 8: Figure S9), which cine oxidation by CuAO are H2O2,NH3,andΔ1-pyrroline; involves the gene copper amine oxidase (CuAO) (Table 4). and Δ1-pyrroline is further catabolized to γ-aminobutyric In plants, wounding of tissue usually results in an increase acid, which subsequently is transaminated and oxidized to in the steady-state levels of copper amine oxidase expres- succinic acid. Thus, in Penicillium copper amine oxidase sion and H2O2 accumulation [32]. Activation of CuAO maycontributetothebalanceofreactiveoxygenspecies during Penicillium attack also may lead to enhanced accu- (ROS) produced in the cell wall extracellular matrix [34]. mulation of H2O2 at the wound site, thereby contributing This specific regulatory mechanism and the molecular to extension of necrotic lesions and extensive plant-cell signals inducing modulation of copper amine oxidase damage [32]. Also, copper amine oxidase may catalyze oxi- in Penicillium during decay highlight the relevance of these dation of the aliphatic diamines putrescine and cadaverine enzymes as an H2O2-delivering system in colonized tissue. Barad et al. BMC Genomics (2016) 17:330 Page 16 of 27

Table 4 Genes involved in each process of cluster 9 GO Term Gene id Genbank transcript id Description Ammonium transmembrane transport PEXP_025350 KGO42664 Ammonium transporter Cellular amino acid metabolic process PEXP_082650 KGO38572 Pyridoxal phosphate-dependent transferase, major region, subdomain 2 PEXP_109810 KGO38703 Glucose-methanol-choline oxidoreductase PEXP_004920 KGO36657 Glutamate synthase, central-N PEXP_053110 KGO42359 Catalase-peroxidase heme PEXP_056170 KGO38539 1-aminocyclopropane-1-carboxylate deaminase Amine metabolic process PEXP_053110 KGO42359 Catalase-peroxidase heme PEXP_056170 KGO38539 1-aminocyclopropane-1-carboxylate deaminase PEXP_000620 KGO44371 Copper amine oxidase, N2-terminal Hydrogen peroxide catabolic process PEXP_053110 KGO42359 Catalase-peroxidase heme

The overrepresentation of glucose-methanol-choline oxido- Interestingly, similar up-regulation of catalase peroxidase reductase- and copper amine oxidase-activated genes in under pathogenic conditions was reported to account for cluster9mayindicatetheircontributiontotheROSand the survival of P. marneffei, an intracellular pathogen that oxidoreductase process at the leading edge of the colony; causes common opportunistic infections in humans, and also, in the same cluster there was activation of the H2O2 of P. simplicissimum, a plant pathogen in which strong catabolic process (Additional file 8: Figure S9) through expression of the catalase peroxidase transcripts may catalase peroxidase, which suggests a hitherto unknown contribute to survival of this fungus in host cells [35]. mechanism of Penicillium survival under oxidative stress. Considered together, the relative expressions of the oxidoreductase genes and copper amine oxidase under fruit pH levels ranging from 3.7 to 4.2, again may indicate the importance of local ammonification, as reported by Barad et al. [8], as a mechanism to induce activation of genes usually overexpressed at pH 7 (Fig. 3). The overrepresentation in cluster 9, of several nitrogen- metabolism-regulating genes, such as glutamate synthase and MepB (Table 4) also is important for nitrogen metabolism in fungi. The ammonia transporter encoded by mepB can lead to an internal/external modulation of ammonia in the hypha, activation of pacC, and high-pH induced genes [36]. Accumulation of glutamate, followed by its transform- ation to glutamine by glutamate synthase activity may be the basis for accumulation of several amino acids, and both stages are activated by ammonia accumulation (Fig. 3). Overall, the nitrogen mobilization promoted by infection couldbeconsideredaspartofa"slash-and-burn"strategy that deprives the pathogen of nutrients and modulates alkaline-expressed genes. Such nutritional and metabolic changes might occur as a differential-attack mechanism promoting pathogen development [37]. One interesting gene also activated in cluster 9 in cellular amino acid metabolism (Additional file 8: Figure S9) is the gene encoding for aminocyclopropane-1-carboxylate deam- Fig. 3 Effects of ammonia and pH on the relative expressions of genes involved in the pathogenicity process. Expressions were inase (ACC) (Table 4). The ACC functions as a deaminase, analyzed of: polygalacturonase (PG), Mep2, Copper amine oxidase degrading aminocyclopropane-1-carboxylate deaminase to (CuAO), ACC deaminase, PacC, and pectate lyase A (PelA). a Samples 2-oxobutyrate and ammonia, which is a precursor of from the leading edge of infection in apples treated with exogenous the plant hormone ethylene. A similar reaction process μ NH4Cl at 0 and 22 M, according to Barad et al. [8]; b Samples grown catalyzing ACC synthase was found in Penicillium digita- at pH 4 or pH 7, for 3 h in shaking secondary media (SM) tum attacking citrus fruits [38, 39], which suggests that Barad et al. BMC Genomics (2016) 17:330 Page 17 of 27

Fig. 4 Heat map of the expression pattern of GMC oxidoreductase gene family. The expression patterns of genes belonging to the GMC oxidoreductase gene family are shown as a heat map obtained with matrix2png software [72]. Gene identity, gene description, and its location in the differentially expressed gene clusters are indicated colonization may be associated with ethylene production to that in vitro at pH 7 (Fig. 2). This cluster shows enrich- and induced tissue senescence. Taken together these re- ment of genes involved in the oxido-reduction process, sults indicate that ROS production by the pathogen may such as cysteine dioxygenase (Table 5, Additional file 9: be accompanied by host tissue induced senescence. Figure S4). Among representatives of this group are the cytochrome P-450 (CytP) monooxygenases, which are Low expression level of fungal genes in colonized apple enzymes that: catalyze conversion of hydrophobic in- tissue and in fungus grown in vitro at pH 7 termediates of primary and secondary metabolic pathways; Group III also includes cluster 4, in which colonized apple detoxify natural and environmental pollutants that are ac- tissue showed under-representation of transcripts similar cumulated during colonization as phytochemical molecules Barad et al. BMC Genomics (2016) 17:330 Page 18 of 27

Table 5 Genes involved in each process of cluster 4 GO Term Gene id genbank transcript id Description Oxidation-reduction process PEXP_035940 KGO40210 Cytochrome P450, E-class, CYP52 PEXP_094610 KGO43560 Multicopper oxidase, type 1 PEXP_069560 KGO47265 Cytochrome P450 PEXP_047900 KGO39614 Cytochrome P450 PEXP_074010 KGO48671 Cytochrome P450 PEXP_065530 KGO46863 Short-chain dehydrogenase/reductase SDR PEXP_073620 KGO48632 Polyketide synthase, enoylreductase PEXP_079160 KGO37900 FAD-linked oxidase, N-terminal PEXP_057820 KGO44289 Cysteine dioxygenase type I PEXP_043230 KGO39482 Isocitrate and isopropylmalate dehydrogenases family PEXP_101170 KGO38159 Oxoglutarate/iron-dependent dioxygenase PEXP_060240 KGO45235 Short-chain dehydrogenase/reductase SDR PEXP_034900 KGO40106 Aldolase-type TIM barrel PEXP_106700 KGO41290 Isocitrate and isopropylmalate dehydrogenases family PEXP_026690 KGO42920 Short-chain dehydrogenase/reductase SDR PEXP_069740 KGO47756 Redoxin PEXP_103230 KGO36363 Cytochrome P450 PEXP_091400 KGO45110 hypothetical protein PEXP_027810 KGO43163 Polyketide synthase, enoylreductase PEXP_023550 KGO42484 Indoleamine 2,3-dioxygenase PEXP_077820 KGO37766 Polyketide synthase, enoylreductase PEXP_043790 KGO39298 Dihydrolipoamide succinyltransferase PEXP_082800 KGO38587 Cytochrome P450 PEXP_043420 KGO39261 Polyketide synthase, enoylreductase PEXP_030200 KGO40444 Cytochrome P450 PEXP_061260 KGO45498 Short-chain dehydrogenase/reductase SDR PEXP_078860 KGO37870 Dimeric alpha-beta barrel PEXP_042100 KGO48408 NADPH-cytochrome p450 reductase, FAD-binding, alpha-helical domain-3 PEXP_037510 KGO35963 Oxoglutarate/iron-dependent dioxygenase PEXP_010380 KGO48989 Aldo/keto reductase PEXP_000410 KGO44350 Acyl transferase/acyl hydrolase/lysophospholipase PEXP_076770 KGO37511 3-oxo-5-alpha-steroid 4-dehydrogenase, C-terminal PEXP_066880 KGO46519 FAD dependent oxidoreductase PEXP_008580 KGO37058 hypothetical protein PEXP_001620 KGO37137 Ubiquinone biosynthesis protein Coq7 PEXP_030860 KGO40271 NADPH-dependent FMN reductase PEXP_107740 KGO41768 Polyketide synthase, enoylreductase PEXP_049110 KGO39496 Monooxygenase, FAD-binding PEXP_017320 KGO46090 Polyketide synthase, enoylreductase Taurine metabolic process PEXP_057820 KGO44289 Cysteine dioxygenase type I PEXP_050220 KGO42072 Acetate/Proprionate kinase Barad et al. BMC Genomics (2016) 17:330 Page 19 of 27

belonging to the polyphenols; and enable fungal growth the various clusters (Fig. 5). CAZymes formed 8.00 % under varied colonizing conditions [40]. They do this by of all the transcripts in cluster 1, with 15, 24, 19, 57, inserting one oxygen atom into the aliphatic position of an and 65 of CAZymes families including auxiliary activity organic substrate, while the other oxygen atom is reduced (AA), carbohydrate-binding modules (CBM), carbohydrate to water. esterases (CE), glycosyltransferases (GT), and glycoside hydrolases (GH), respectively. In cluster 2 they formed 5.57 % Carbohydrate active enzymes (CAZy) cluster distribution of all the transcripts with 2, 3, and 8 of CAZymes families The diverse complex carbohydrates that contribute to including carbohydrate esterases, glycosyltransferases Penicillium maceration capabilities are controlled by a (GT), and glycoside hydrolases (GH), respectively. In clus- panel of enzymes involved in their assembly (glycosyltrans- ter 3 they formed 17.92 % of all the transcripts, with 24, 74, ferases) and breakdown (glycoside hydrolases, polysacchar- 28, 149, and 123 of CAZymes families including auxiliary ide lyases, carbohydrate esterases), which collectively are activity (AA), carbohydrate-binding modules (CBM), designated as Carbohydrate-Active enZymes (CAZymes) carbohydrate esterases (CE), glycosyltransferases (GT), [41]. In plant pathogens CAZymes promote synthesis, deg- and glycoside hydrolases (GH), respectively. In cluster radation, and modification of carbohydrates that play im- 4 they formed 14.46 % of all transcripts, with 2, 3, 4, portant roles in the breakdown of plant cell walls and in 15, and 11 of CAZymes families including including host/pathogen interactions [42]. Penicillium uses these en- auxiliary activity (AA), carbohydrate-binding modules zymes to macerate the colonized tissue by degradation of (CBM), carbohydrate esterases (CE), glycosyltransfer- complex carbohydrates of the hosts to simple monomers ases (GT), and glycoside hydrolases (GH), respectively. that can be utilized as nutrients [43, 44]. In cluster 5 they formed 22.61 % of all transcripts, with The CAZymes analysis toolkit (CAT) [45] was used to 7, 11, 11, 5, 23, and 52 of CAZymes families including identify 771 putative CAZymes in P. expansum within auxiliary activity (AA), carbohydrate-binding modules

Fig. 5 Comparison of the CAZyme repertories identified in each cluster of co-expressed genes. Enzyme families are represented by their class (GH-glycoside hydrolases; GT-glycosyltransferases; PL-polysaccharide lyases; CE-carbohydrate esterases; and CBM-chitin-binding modules) and family number according to the Carbohydrate-Active Enzyme Database. Abundance of the various enzymes within a family is represented on a color scale from 0 (dark blue) to 150 occurrences (dark red) per cluster Barad et al. BMC Genomics (2016) 17:330 Page 20 of 27

(CBM), carbohydrate esterases (CE), polysaccharide lyases Table 6 Apple up-regulated pathways by infection with P. (PL), glycosyltransferases (GT), and glycoside hydrolases expansum (GH), respectively. In cluster 6 they formed 29.16 % of all Pathway name p value (adjusted) transcripts, with 2, 3, 2, 5, and 9 of CAZymes families in- Jasmonic acid biosynthesis 5.73E-11 cluding auxiliary activity (AA), carbohydrate-binding mod- Mevalonate pathway 9.12E-08 ules (CBM), PL, glycosyltransferases (GT), and glycoside Flavonoid biosynthesis 7.62E-05 hydrolases (GH), respectively. In cluster 7 they formed Superpathway of geranylgeranyldiphosphate 7.62E-05 22.61 % of all transcripts, with 4 of CAZymes families in- biosynthesis I (via mevalonate) cluding glycosyl hydrolases. In cluster 8 they formed Glutathione-mediated detoxification 0.00171 12.06 % of all transcripts, with 2 and 5 of CAZymes families including carbohydrate esterases (CE) and glycoside Trans,trans-farnesyl diphosphate biosynthesis 0.0032 hydrolases (GH), respectively. In cluster 9 they formed Glutamate dependent acid resistance 0.0032 16.67 % of all transcripts, with 2 of CAZymes families Chorismate biosynthesis 0.00325 including each of auxiliary activity (AA) and glycoside Amygdalin and prunasin degradation 0.00988 hydrolases (GH) (Fig. 5). Salicylate biosynthesis 0.01194 Carbohydrate esterases, glycoside hydrolases, and DIMBOA-glucoside degradation 0.01415 polysaccharide lyases are associated with the ability to β utilize the diversity of carbohydrates present in the en- -Alanine biosynthesis II 0.01456 vironment and within host fruits. Glycosyltransferases Acetate formation from acetyl-CoA II 0.01456 are mainly involved in the basal activities of fungal Pyruvate fermentation to acetate III 0.01456 cells, e.g., fungal-cell-wall synthesis, glycogen cycle, Glutamate degradation III (via 4-aminobutyrate) 0.01693 and trehalose cycle [41, 44, 46]. The wide occurrence Superpathway of phenylalanine and tyrosine 0.01693 of CAZymes in the various Penicillium clusters, taken biosynthesis together with their importance in degradation of the Phospholipid desaturation 0.03878 plant cell wall, indicates their basic contribution to Glycolipid desaturation 0.03878 colonization of the host fruit. 13-LOX and 13-HPL pathway 0.04864 Differentially expressed genes in Penicillium expansum- and Divinyl ether biosynthesis II (13-LOX) 0.04864 Colletotrichum gloeosporioides-infected apple compared Superpathway of phenylalanine, tyrosine, and 0.04864 with healthy tissue tryptophan biosynthesis In order to analyze the effect of Penicillium expansum The analysis was performed with MetGenMap infection during acidification on the host (apple tissue) response, we used RNAseq to analyze the differentially expressed genes induced by P. expansum infection. Overall, via the octadecanoid pathway [49]. Biosynthesis of jasmo- in the P. expansum-infectedappletissuewefound4,292 nates starts with oxygenation of linolenic acid, which is differentially expressed genes with FDR threshold < 0.001 thought to be released from membrane lipids through the and with expression levels increasing or decreasing by a fac- action of a lipase, followed by several oxidation processes. tor greater or less than 8, respectively, i.e., greater or less This activation may indicate that there is a host response than +3 or −3, respectively, on a logarithmic (base 2) scale. prior to fungal maceration induced by Penicillium,andthis We found 2,427 up-regulated and 1,865 down-regulated supposition is supported by findings that fruits pretreated genes in colonized apple tissue. Analysis of the enriched with JA and related compounds showed enhanced resist- pathways with MatGeneMap [47] revealed 21 significantly ance to pathogens [50]. up-regulated pathways (Table 6) and 21 down-regulated The second process that was activated was the mevalo- ones (Table 7) with FDR < 0.05. Among the significantly nate pathway, which produces isoprenoids that are vital for up-regulated processes induced by Penicillium in apple tis- diverse cellular functions; these isoprenoids include sterols, sue were: the jasmonic acid (JA), the mevalonate, and the carotenoids, chlorophyll, plant hormones, and defense iso- flavonoid biosynthesis pathways, and the geranyl geranyldi- prenoids. The penetration of Penicillium probably induces phosphate biosynthesis I super pathway. Among the down- defense isoprenoids such as were found in damaged plant regulated pathways were the glycogen biosynthesis I and leaves [51]. These comprise a wide variety of defense- the starch biosynthesis pathways. Jasmonic acid is a lipid- related genes, including those that activate biosynthesis of derived signaling compound involved in regulation of di- JA and ethylene, as a possible response to pathogen pene- verse processes in plants such as fruit ripening, root growth, trations, as was reported for Botrytis cinerea [52]. qRT-PCR tendril coiling, senescence, and resistance to pathogens analysis of the ethylene-responsive transcription factor [48]; JA and related compounds are synthesized in plants 2-like showed a significant induction of this gene in Barad et al. BMC Genomics (2016) 17:330 Page 21 of 27

Table 7 Apple down-regulated pathways by infection with the apple tissue as a result of the Penicillium infection P. expansum (Table 8). Pathway name p value (adjusted) The third up-regulated process was related to biosyn- Glycogen biosynthesis I (from ADP-D-Glucose) 9.064E-08 thesis of flavonoids, which are linked to fungal potential Starch biosynthesis 6.194E-07 cytotoxicity and capacity to interact with enzymes through protein complexation [53]. Some flavonoids provide stress C4 Photosynthetic carbon assimilation cycle 0.0002403 protection, for example, by acting as scavengers of free UDP-galactose biosynthesis (salvage pathway 0.0008945 from galactose using UDP-glucose) radicals such as reactive oxygen species (ROS), as reported by Falcone Ferreyra et al. [53]. During P. expansum patho- Lipoate biosynthesis and incorporation I 0.0019429 genicity, the fungus produces gluconic acid, with H2O2 as Methylerythritol phosphate pathway 0.0031538 a by-product. The host reacts to ROS by activating the Acyl carrier protein metabolism 0.0034133 flavonoids biosynthesis pathway and thereby initiating a Trans-lycopene biosynthesis 0.0059809 resistance response to fungal penetration. Production Fatty acid biosynthesis initiation I 0.0070497 of secondary metabolites such as anthocyanins, isofla- Pyridoxal 5'-phosphate biosynthesis 0.0087438 vonoids, and flavonol glycosides may contribute to this resistance. Plants such as Arabidopsis respond to the Superpathway of pyridoxal 5'-phosphate 0.0132283 biosynthesis and salvage combination of biotic, i.e., bacterial, and abiotic, e.g., UV-B radiation, stresses through synthesis of defense- Xylitol degradation 0.0161255 related compounds such as phytoalexins and lignin, which Starch degradation 0.0173149 serve as structural barriers that restrict the spread of patho- Heme biosynthesis I 0.0178181 gens. These responses modify the expression of genes in- Heme biosynthesis from uroporphyrinogen I 0.0195451 volved in the production of protective metabolites such as Colanic acid building blocks biosynthesis 0.0199615 flavonols [53]. This behavior suggests that there is a signifi- Xylose degradation I 0.0284483 cant apple response to Penicillium penetration under these susceptible conditions. This activation matches the fourth Sucrose biosynthesis 0.0303525 activated pathway of P. expansum colonization, in which 5-Aminoimidazole ribonucleotide biosynthesis II 0.0332884 the geranyl-geranyl diphosphate-mediated processes are ac- Biotin-carboxyl carrier protein 0.0357646 tivated, probably in biosynthesis of essential compounds Methanol oxidation to formaldehyde 0.0437164 such as chlorophylls, carotenoids, tocopherols, plastoqui- The analysis was performed with MetGenMap nones, and gibberellins, but mainly in production of a variety of secondary metabolites. All of which indicates thesignificantresponseofapplefruitsincopingwith the colonization process.

Table 8 Relative expression (Pe/Cg) of selected genes in apple tissue infected with P. expansum or C. gloeosporioides Apple down-regulated genes Apple (Pe)/apple (Cg) Apple up-regulated genes Apple (Pe)/apple (Cg) Programmed cell death protein 4-like 0.025/0.112 = 0.223 Histone deacetylase hdt3-like 0.70/1.21 = 0.583 Auxin-repressed kda isoform x1 0.002/0.009 = 0.222 Phenylalanine ammonia-lyase 1 9.41/3.38 = 2.781 ap2-like ethylene-responsive transcription 0.015/0.063 = 0.238 Chalcone synthase 16.33/24.23 = 0.670 factor at2g41710 isoform x2 udp-glucose:glycoprotein glucosyltransferase 0.104/0.291 = 0.357 Peroxidase 47 98.83/10.55 = 9.368 Senescence-associated carboxylesterase 101-like 0.079/0.192 = 0.411 Respiratory burst oxidase homolog 185.14/24.15 = 7.664 protein d-like 1-Aminocyclopropane-1-carboxylate oxidase 1 0.034/0.064 = 0.544 Lipoxygenase 490.05/205.74 = 2.381 Anthocyanidin 3-o-glucosyltransferase 5-like 0.007/0.041 = 0.169 Indole-3-acetic acid-induced protein arg2 9.97/6.49 = 1.535 Expansin 1 0.002/0.040 = 0.067 Zinc finger an1 domain-containing 17.66/17.78 = 0.993 stress-associated protein 12-like ap2-like ethylene-responsive transcription factor 0.012/0.045 = 0.282 NADPH–cytochrome p450 reductase 8.18/4.04 = 2.022 at2g41710 isoform x2 isoform x2 Lysine-specific histone demethylase 1 homolog 1-like 0.075/0.192 = 0.394 Ethylene-responsive transcription 33.81/33.83 = 0.999 factor 2-like Serine threonine-protein kinase-like 11.12/1.83 = 6.049 protein ccr4 The relative expression of the apple genes was compared with healthy tissue Barad et al. BMC Genomics (2016) 17:330 Page 22 of 27

Two significantly down-regulated processes that modu- colonization similar to those obtained under in vitro growth late carbohydrate metabolism were found: glycogen biosyn- conditions at pH 7. These three main trends indicate the thesis I from ADP-D-glucose, and starch biosynthesis. existence of pH-regulated genes, expressed at pH 4 and Glycogen and starch are both multibranched polysaccha- pH 7 that may contribute to P. expansum colonization. rides of glucose that serve as a means of energy storage for One of the key processes overrepresented in cluster 7 at fungi; their downregulation may indicate their importance pH 4 was high expression of glutamate decarboxylase as energy stores that rapidly can be mobilized from the (GAD). One of the key products of this enzyme-GABA- cytosol/cytoplasm and that perform an important function largely originates from decarboxylation of L-glutamate [54], during glucose consumption by Penicillium during the and it had been found to function in communication acidification process [7]. Down-regulation of these pro- between tomato (Lycopersicon esculentum var. commune cesses probably results from catabolism of the substrates Bailey) plants and the fungus Cladosporium fulvum [55]. during attack by P. expansum and strongly supports previ- Hyphae of C. fulvum are restricted to the apoplast, there- ous findings of Prusky et al. [3], Hadas et al. [4], and Barad fore the fungus is dependent on the contents of the et al. [7] that indicate acidification of the tissue as a factor apoplastic nutrients. During infection, the GABA concen- in pathogenicity of P. expansum. tration in the apoplast increased from about 0.8 mM to 2– In order to analyze the responses of apple fruits to 3 mM; this can be attributed to stimulation of glutamate fungal acidification or alkalization, the differential ex- dehydrogenase activity by decreased pH and increased pressions of specific genes induced during colonization cytosolic calcium, which are associated with pathogen at- by Penicillium were compared to those expressed during tack [56]. In the present study, similar conditions were colonization of the same host by Colletotrichum. For present in the Penicillium/apple interactions, where acidifi- that purpose, the relative expressions of 21 specific key cation by GLA accumulation may enhance the consump- genes, of which 10 were up-regulated and 11 down- tion of this amino acid. regulated by Penicillium (Table 8), were compared in Accumulation of ammonia, induced under the limited- apple RNA extracted from the leading edges of tissue nutrient conditions present at the edge of the decaying colonized by the two respective pathogens. Genes that tissue, may contribute to gene expression, either by alka- were up-regulated in the fruit showed the same response lization or by direct induction of ammonia during gene pattern when fruits were colonized by either Penicillium activation [8]. Modulation of ammonia levels by MepB or Colletotrichum, but the relative expression obtained transporters and/or the amine metabolic process (both in Penicillium-inoculated apples was always significantly overrepresented at pH 7 and decayed tissue in cluster 9) higher than that in Colletotrichum-inoculated apples: the may contribute to the alkalizing effects and pH increase. ratio of P. expansum: C. gloeosporioides relative expression Under these pH conditions also an over-representation responses ranged from 0.58 to 9.36. This indicates a of glucose-methanol-choline (GMC) oxidoreductase was stronger host response to Penicillium colonization than observed. GMC shows similar activity to the GOX of P. to Colletotrichum colonization. Analysis of the down- expansum that contributed to oxidation and reduction regulated genes also showed similar patterns of host re- processes [29] by providing the H2O2 required by ligni- sponses to the two respective pathogens, but lower ratios nolytic peroxidases in Pleurotus species [57]. This com- of P. expansum: C. gloeosporioides relative expressions, bination of GMC genes [58], described here for the first ranging from 0.067 to 0.54, which indicates that, although time in P. expansum under alkaline conditions, indicates the response patterns were similar, the alkalizing pathogen activity of new undescribed mechanisms that contribute induced lower expression levels. These results indicate to cell-wall degradation by generation of H2O2 during that host responses to Penicillium and to Colletotrichum fungal colonization at pH 7. attack did not show opposite senses of gene modulation, In addition, the copper amino oxidase (CuAO) con- as might be expected from their contrasting pH modula- tributes to the enhanced accumulation of H2O2 at the tion directions, but only reduced expression levels in the wound site which, in turn, contributes to the extended Colletotrichum-inoculated apples. necrotic lesions and extensive plant cell damage [32]. The CuAO catalyzes oxidation of aliphatic diamines of Conclusions the primary amino groups that contribute to H2O2 and Overall, gene-expression profiling of pH-dependent genes NH3 accumulation, which emphasizes the relevance of of P. expansum during colonization of apple fruits re- the H2O2-delivering system in colonized tissue [33]. This vealed three major effects: (1) a pattern of high gene over-representation of GMC and CuAO indicates the expression during colonization that had no connection contribution to ROS production and oxidoreductase with pH response; (2) gene expression patterns during processes at the leading edge of the developing colony, colonization similar to those obtained under in vitro growth and attributes the presence of fungal catalase peroxidases conditions at pH 4; and (3) gene expression patterns during the need to protect colonizing hyphae. Barad et al. BMC Genomics (2016) 17:330 Page 23 of 27

These oxidative processes were concurrent with acti- water supplemented with 0.01 % (v/v) Tween 80 (Sigma- vation of Cyt P-450 monoxygenase-in group 3, cluster Aldrich, Copenhagen, Denmark). Cells were visualized 4-which catalyzes the conversion of hydrophobic inter- with a model BX60F-3 microscope and a model SZ-60 mediates of primary and secondary metabolic pathways, stereoscope (both from Olympus America Inc., Melville, thereby detoxifying natural and environmental pollutants NY, USA) and counted with a hemocytometer (Brand, and allowing fungi to grow under difficult oxidative condi- GMBH, Werheim, Germany). tions [40], which accounts for the activation of genes coding for proteases, cell-wall-related enzymes, redox Assay for colonization and disease development homoeostasis, and detoxification processes that are 'Golden Delicious' apples were inoculated by placing − expressed during the infection process. 5 μL of a conidial suspension containing 106 spores mL 1 Exploiting these strong oxidative conditions during its on each of six 2-mm-deep, 2-mm-diameter wounds necrotrophic development, the fungus further advances the spaced evenly in a circle around the upper part of the stem colonization process by activation of aminocyclopropane-1- end of the fruits. Following inoculation, the fruits were in- carboxylate deaminase (ACC) (cluster 9), a precursor of the cubated for 5 days at 25 °C in covered plastic containers plant hormone ethylene. A similar reaction process catalyz- containing wet paper towels. Samples of healthy tissue ing ACC synthase was found when P. digitatum attacked and of the leading edge of each wound were collected and citrus fruits [38, 39], which indicates that the colonization frozen with liquid nitrogen for subsequent RNA analysis. process involves induction of senescence of the colonized tissue, in conjunction with the strong oxidative process. In vitro procedure of P. expansum − Out of the 771 putative CAZymes identified in P. Fungal spores were inoculated at 106 spores mL 1 into expansum, eight were found to be expressed in clusters 40 mL of a primary medium, i.e., glucose minimal 7 and 9, which were the most important clusters modu- medium, in 125-mL flasks containing (per liter) 10 g su- lating pathogenicity (Fig. 5). Given the importance of crose, 5 g yeast extract (Difco Laboratories, MD, USA), CAZymes during cell-wall degradation, it is clear that 50 mL nitrate salts, and 1 mL trace elements, at pH 4.5. they contribute strongly to the apple maceration process. The cultures were incubated at 25 °C with shaking at The present transcriptome analysis showed induced am- 150 rpm for 48 h. Cultures were harvested by vacuum monification, and strong oxidative and senescence pro- filtration through a sterile Büchner funnel fitted with a cesses, accompanied by strong activation of pectolytic Whatman number 1 filter paper, and the remaining my- enzymes, all of which indicate the pH dependence of the celia were washed twice with 50 mL of sterile distilled tools used by the pathogen to colonize the environment. water. The washed mycelia were resuspended in 50 mL All these induced metabolic changes indicate the signifi- of 0.2 M phthalate-buffered or phosphate-buffered liquid cant role of pH modulation in the pathogenicity of P. secondary medium (SM) at pH 4 or pH 7, respectively. expansum in fruits. This SM contained (per liter) 60 g sucrose, 7 g NaNO3, Analysis of the fruit transcriptome suggested that even 3 g tryptone (Difco Laboratories, MD, USA), 1 g KH2PO4, if apple fruits are susceptible to Penicillium, the fungus 0.5 g MgSO4 ·7H2O, and 0.5 g KCl. The cultures were in- activates significant gene processes related to fruit resist- cubated at 25 °C on a rotating shaker at 150 rpm for 0.5, ance. This may indicate that the necrotrophic maceration 1, 3, 10, or 24 h. A sample of mycelia from the cultures of the tissue occurs, not as a senescence response, but as was collected into a 1.5-mL Eppendorf at each time point, an active fungal process that supports its pathogenicity. and the mycelia were frozen with liquid nitrogen for RNA Also interesting is the stronger response of Penicillium extraction. than of Colletotrichum, which indicates that the pH ad- justment effected by the fungus did not affect the pattern RNA extraction but the level of host response. At the same time, these RNA was extracted from the in vitro samples with the findings indicate that the fungal matching to the host is SV Total RNA Isolation kit (Promega, Madison,WI, the main factor activating the maceration process. USA). Purity of the extracted RNA was assayed with an ND-1000 spectrophotometer (NanoDrop Technologies Methods Inc., Wilmington, DE, USA), and the extracts were stored Fungal strains and culture conditions at −80 °C pending further analysis. The WT P. expansum isolate Pe-21 was obtained from Total RNA of apple fruits was extracted according to decayed apples (Malus domestica cv. Golden Delicious) Yang et al. [59], with minor changes: aliquots were taken purchased from a local market in Israel [4]. Cultures from pooled samples from the leading edges of the six were grown at 27 °C in the dark, and maintained on inoculation areas of each apple. The samples were PDA plates (Difco, MD, USA) unless otherwise indicated. ground to a fine powder in liquid nitrogen and trans- Conidia were harvested with 10 mL of sterile distilled ferred into 50-mL centrifuge tubes with 10 mL of CTAB Barad et al. BMC Genomics (2016) 17:330 Page 24 of 27

RNA extraction buffer comprising 100 mM Tris-borate in pH 4 for 3 h with 21,506,394 and 26,786,825 reads, pH 8, 2 M NaCl, 25 mM ethylenediaminetetraacetic acid respectively; 2) duplicates of in-vitro P. expansum myce- (EDTA) pH 8, 2 % (w/v) CTAB, 2 % (w/v) polyvinylpoly- lia in pH 7 for 3 h with 16,745,224 and 32,103,824 reads, pyrrolidone, and 2 % (v/v) β-mercaptoethanol. The mix- respectively; 3) duplicates of pooled samples from all ture was shaken for 3 min and then incubated at 65 °C time points during in vitro exposure in medium at pH 4, for 15 min. Samples were extracted twice with an equal with 19,665,360 and 14,581,483 reads, respectively; (4) volume of 24:1 (v:v) chloroform:isoamyl alcohol, and the duplicates of pooled samples from all time points during phases were separated by centrifugation at 10,000 × g for in vitro exposure in medium at pH 7, with 17,324,922 10 min. Following centrifugation, LiCl was added to a and 19,406,152 reads, respectively; (5) duplicates of leading final concentration of 2.5 M and RNA was allowed to edge samples of inoculated apple tissue with 253,299,784 precipitate overnight at 4 °C. The precipitated RNA was and 199,419,262 reads; (6) one sample of healthy apple cv. pelleted at 4 °C for 30 min at 10,000 × g, washed with 'Golden Delicious' tissue, with 31,719,018 reads. 70 % ethanol, and resuspended at 65 °C for 3 min in The datasets are available at the NCBI Sequence Read SSTE buffer comprising 10 mM Tris pH 8, 1 M NaCl, Archive (SRA) under accession number SRP071104. 1 mM EDTA pH 8, and 0.5 % (w/v) SDS. Samples were The Bowtie2 softweare [14] was used to align the RNA- extracted with equal volumes of 24:1 (v:v) chloroform:i- seq outputs against the transcriptome of P. expansum.The soamyl alcohol, and with equal volumes of 24:1:25 (v:v:v) libraries were aligned against the P. expansum genome chloroform:isoamyl alcohol:water-saturated phenol, and (downloaded from NCBI Accession no. JQFX00000000.1). the phases were separated by centrifugation at 10,000 × g The apple samples were also aligned against the Malus × for 10 min. The RNA was ethanol-precipitated over- domestica. Whole Genome v1.0 that was downloaded from night, and resuspended in diethyl-pyrocarbonate-treated GDR (Genome Database for Rosaceae) [60]. RSEM soft- water. The RNA was further treated with Turbo DNAse ware [61] was used for transcript quantification of the (Ambion, Austin, TX, USA). RNAseq data and then the edgeR package [62] was used to calculate differentially expressed genes. The genes of P. expansum were annotated by using Preparations of libraries BLASTx [63] against the non-redundant NCBI protein A 500-ng of total RNA from 11 samples was processed database, after which their GO term [64] was assigned with the TruSeq RNA Sample Preparation Kit v2 (RS- by combining BLASTx data and interproscan analysis 122-2001) (Illumina, San Diego, CA, USA). Libraries [65] by means of the BLAST2go software pipeline [16]. were evaluated with the Qubit and TapeStation software GO-enrichment was analysed by using Fisher’s Exact (Agilent Technologies, Palo Alto, CA, USA), and se- Test with multiple testing correction of FDR [15]. Heat- quencing libraries were constructed with barcodes to map and clustering of the genes were visualized by using enable multiplexing of pools of the eight in vitro sam- the R software ggplots2 package [66]. We used a thresh- ples-duplicates of pH 4 3 h, pool pH 4, pH 7 3 h and pool old of FDR < 0.05 [15] and the criterion that expression pH 7-in one lane. The results showed 14.5–32.1 million level increased or decreased by a factor greater or less than single-end 50-bp reads. 2, respectively, i.e., greater than or less than +1 or −1, re- Three individual lanes were used for the in vivo samples spectively, on a logarithmic (base 2) scale. The normalized and yielded 199.5–253.3 million single-end, 100-bp reads expression value was centered and log 2 transformed for from the duplicates of the leading edges of infected apple visualization purposes with a script taken from trinity tissue and 31.7 million single-end 100-bp reads from pipeline [67]. The CAZY analysis was done with the healthy apple tissue. All samples were sequenced on a CAZymes Analysis Toolkit (CAT) [45]. A heat map of HiSeq 2500 The transcriptome of the healthy apple tissue Cazymes in each cluster was plotted by using the R pack- and the leading edge of infected tissue were sequenced age pheatmap [68]. with Trueseq protocols at the Genome Center of the Life Sciences and Engineering Faculty, Technion-Israel Inst. Technology, Haifa, Israel. Ammonia atmosphere analysis To create an ammonia vapor atmosphere, 250-mL aliquots Bioinformatic analysis of RNAseq Data of 5 N NaOH with 0 or 50 μMofNH4Cl were placed in Three libraries with total single-end, 100-nucleotides- closed containers for 24 h before use, after which, five apple long RNA-seq reads were generated from the in vivo fruits were placed inside each container for 5 days. Concen- samples and eight libraries with 50-nucleotides-long trations of 0 and 50 μMNH4Cl in NaOH solutions resulted total single-end RNA-seq reads were generated from the in detected ammonia concentrations of 0 and 22 μM, re- in vitro samples. The libraries contained the following spectively. All presented gene-expression results are relative sequences: 1) duplicates of in-vitro P. expansum mycelia to 0 μMNH4Clin5NNaOH. Barad et al. BMC Genomics (2016) 17:330 Page 25 of 27

Gene-expression analysis by qRT-PCR Additional file 4: Figure S6. GO-enriched terms of genes in cluster 6. Real-time qPCR was performed with the StepOnePlus GO enrichment was calculated by using Fisher's Exact Test with blast2go System (Applied Biosystems, Grand Island, New York, software [18]. All Penicillium expansum genes served as background for μ the calculations. The percentages of the GO term in all P. expansum USA). PCR amplification was performed with 3.4 Lof genes are marked in red, and the percentages of the GO terms in the cDNA template in 10 μL of reaction mixture containing gene clusters are marked in blue. (JPG 159 kb) 6.6 μL of the mix from the SYBR Green Amplification Additional file 5: Figure S7. GO-enriched terms of genes in cluster 7. Kit (ABgene, Surrey, UK) and 300 nM of primers; GO enrichment was calculated by using Fisher's Exact Test with blast2go software [18]. All Penicillium expansum genes served as background for Additional file 10: Tables S1 and Additional file 11: the calculations. The percentages of the GO term in all P. expansum TableS2(seeSupportingInformation)listtheforward genes are marked in red, and the percentages of the GO terms in the and reverse primers for each of the indicated genes. gene clusters are marked in blue. (JPG 164 kb) The PCR was carried out as follows: 10 min at 94 °C, Additional file 6: Figure S5. GO-enriched terms of genes in cluster 5. GO enrichment was calculated by using Fisher's Exact Test with blast2go and 40 cycles of 94 °C for 10 s, 60 °C for 15 s, and 72 °C software [18]. All Penicillium expansum genes served as background for for 20 s. The samples were subjected to melting-curve the calculations. The percentages of the GO term in all P. expansum analysis: efficiencies were close to 100 % for all primer genes are marked in red, and the percentages of the GO terms in the gene clusters are marked in blue. (JPG 159 kb) pairs, and all products showed the expected sizes of 70 to Additional file 7: Figure S8. GO-enriched terms of genes in cluster 8. 100 bp. All of the samples were normalized to 28S expres- GO enrichment was calculated by using Fisher's Exact Test with blast2go sion levels, and the values were expressed as the change software [18]. All Penicillium expansum genes served as background for (increasing or decreasing) of levels relative to a calibrator the calculations. The percentages of the GO term in all P. expansum genes are marked in red, and the percentages of the GO terms in the sample. Results were analyzed with the StepOnePlus soft- gene clusters are marked in blue. (JPG 164 kb) ware v.2.2.2 (Applied Biosystems, Grand Island, New Additional file 8: Figure S9. GO-enriched terms of genes in cluster 9. York, USA). Relative quantification was performed by the GO enrichment was calculated by using Fisher's Exact Test with blast2go software [18]. All Penicillium expansum genes served as background for ΔΔCT method [69]. The ΔCT value was determined by the calculations. The percentages of the GO term in all P. expansum subtracting the CT results for the target gene from those genes are marked in red, and the percentages of the GO terms in the for the endogenous control gene-18S for apple analysis gene clusters are marked in blue. (JPG 196 kb) and 28S for P. expansum analysis-and normalized against Additional file 9: Figure S4. GO-enriched terms of genes in cluster 4. GO enrichment was calculated by using Fisher's Exact Test with blast2go the calibration sample to generate the ΔΔCT values. Each software [18]. All Penicillium expansum genes served as background for experiment was performed in triplicate, and three different the calculations. The percentages of the GO term in all P. expansum biological experiments were conducted. One representative genes are marked in red, and the percentages of the GO terms in the ΔΔ set of results is presented as mean values of 2- CT ±SEfor gene clusters are marked in blue. (JPG 123 kb) each treatment. Additional file 10: Table S1. Fungal primers used in this study. (DOCX 13 kb) Additional file 11: Table S2. Apple primers used in this research. (DOCX 15 kb) Availability of data and material All data presented in this manuscript were deposited in Competing interests the NCBI Sequence Read Archive (SRA) database under The authors declare that they have no competing interests. accession number SRP071104. Authors’ contributions SB and NS analyzed the data and contributed to the chemical analyses; DV, Additional files AD, and NG contributed to specific experiments; AS and DP contributed to writing the manuscript and final analysis of the data. All authors read and approved the final manuscript. Additional file 1: Figure S1. GO-enriched terms of genes in cluster 1. GO enrichment was calculated by using Fisher's Exact Test with blast2go Acknowledgements software [18]. All Penicillium expansum genes served as background for We acknowledge the financial support for D.P. from the US/Israel Binational the calculations. The percentages of the GO term in all P. expansum Agricultural R&D (BARD) fund; project I-IS-4773-14. We appreciate the genes are marked in red, and the percentages of the GO terms in the contributions of the Bioinformatics units of the Weizmann Institute and gene clusters are marked in blue. (JPG 231 kb) the Technion – Israel Institute of Technology, in preparing libraries and Additional file 2: Figure S2. GO-enriched terms of genes in cluster 2. providing sequencing services. GO enrichment was calculated by using Fisher's Exact Test with blast2go software [18]. All Penicillium expansum genes served as background for Funding the calculations. The percentages of the GO term in all P. expansum We acknowledge funding by the US/Israel Binational Agricultural Research genes are marked in red, and the percentages of the GO terms in the Fund, BARD, IS-4773-14. SB was funded by a scholarship of the Volcani gene clusters are marked in blue. (JPG 159 kb) Center, Israel. Additional file 3: Figure S3. GO-enriched terms of genes in cluster 3. GO enrichment was calculated by using Fisher's Exact Test with blast2go Author details 1Department of Postharvest Science of Fresh Produce, Agricultural Research software [18]. All Penicillium expansum genes served as background for 2 the calculations. The percentages of the GO term in all P. expansum Organization, the Volcani Center, Bet Dagan 50250, Israel. Department of Plant Pathology and Microbiology, Robert H. Smith Faculty of Agriculture, genes are marked in red, and the percentages of the GO terms in the gene clusters are marked in blue. (JPG 212 kb) Food and Environment, Hebrew University of Jerusalem, Rehovot 76100, Israel. 3Department of Plant Pathology and Weed Research, ARO, the Volcani Barad et al. BMC Genomics (2016) 17:330 Page 26 of 27

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